Gallick 1 10 07

Cancer Cell Signaling-1: 

HGF Cancer Cell Signaling-1 V. Levin, Cancer Treat Res PDGF VEGF

Problems:: 

Problems: What regulates specificity? Growth factors? Growth Factor Receptors? Overlapping downstream “relays” Transcription? Chromatin Structure? All of the Above? What should be targeted for therapy? Is it surprising that single STIs fail?

Cancer Cell Signaling 2: Extracellular Matrix : 

Cancer Cell Signaling 2: Extracellular Matrix V. Levin Cancer Treat Res

Problems:: 

Problems: Cell-Cell; Cell Extracellular Matrix (ECM) are full participants in development, proliferation, and homeostasis Alterations in these pathways are common in most tumors Alterations in these pathways may not affect proliferation, primarily Cross-talk with receptor-mediated pathways occurs Must these pathways be inhibited for effective therapy?

History: 

History How do we know these are pathways? Yeast, drosophila and man all have them Why are the “intermediates” keys in cancer cell signaling? Intermediates regulate common cellular processes Ironically, alterations are not (immediately) lethal The field originates, and many of our concepts are derived from the study of retroviruses

RETROVIRUSES (RNA TUMOR VIRUSES): A Short History: 

RETROVIRUSES (RNA TUMOR VIRUSES): A Short History Non-Transforming (Leukemia Viruses) Ellerman and Bang, 1908 Isolated from nearly every vertebrate Some are common causes of leukemia FeLV (Feline Leukemia Virus) HTLV (Human T Cell Leukemia Virus)-endemic in the Carribbean and parts of Japan Induce leukemia after long latency periods Do not “transform” tissue culture cells

Peyton Rous: 

Peyton Rous Reported his namesake Rous Sarcoma Virus In 1911 Nobel Prize: 1966

Retrovirus History, cont.: 

Retrovirus History, cont. Transforming (Sarcoma Viruses) Peyton Rous, 1911 -isolated in an elegant series of experiments in which he demonstrated cell free lysates from chickens could induce sarcomas in other chickens Induce a variety of solid tumors and some hematapoetic tumors About 25 odd viruses have been isolated Transform cultured cells Usually contain a single gene responsible for all the tumorigenic properties

Genome of Leukemia Viruses: 

Genome of Leukemia Viruses GENOME: +-stranded RNA STRUCTURE: 2 identical 35S subunits Genetic Organization: gag (group specific antigens): encode proteins which are primarily structural pol (polymerase) encodes RNA-Dependent DNA polymerase (reverse transcriptase) LTR LTR gag pol env

RNA-directed DNA Polymerase(reverse transcriptase): 

RNA-directed DNA Polymerase (reverse transcriptase) David Baltimore Howard Temin Nobel Prize: 1975

Genetic Organization of Leukemia Viruses (cont.): 

Genetic Organization of Leukemia Viruses (cont.) pol (cont.)- encodes r.t. encodes PROTEASE-cleaves structural proteins encodes INTEGRASE-involved in integration of cDNA into chromosoma DNA env (envelope)- encodes 2 proteins, including a glycoprotein involved in attachment LTR (Long Terminal Repeat)- promoter; enhancer for viral replication LTR LTR gag pol env

Genetic Organization of SarcomaViruses: 

Genetic Organization of SarcomaViruses Rous Sarcoma Virus: replication competent, transformation competent, i.e. rapidly induces metastatic sarcomas in infected birds Other “Sarcoma Viruses”: LTR LTR gag pol env src gag onc LTR LTR truncated replication sequences from parental leukemia virus viral oncogene (v-onc) v-src replication- defective viruses with oncogene acquired at the expense of replication genes

Human Immunodeficiency Virus (HIV): 

Human Immunodeficiency Virus (HIV) Has gag, pol, and env genes Does not have a transforming gene Has other genes invlved in transcriptional enhancement and viral replication, e.g, tat, ars, etc.

DNA “Tumor Viruses”: 

DNA “Tumor Viruses” Hepatitis viruses HPV Vaccine makes big news in 2006 for cervical cancer Why has it been so difficult to develop HIV vaccines? Instability of the genome is a critical reason

Sarcoma Viruses: 

Sarcoma Viruses Have provided exquisite genetic tools for the study of the process of malignant transformation Have provided a major source of oncogenes for subsequent study* Rare existence in nature limits their usefulness *SEE NEXT

A Small List Of Transforming Retroviruses: 

A Small List Of Transforming Retroviruses VIRUS Rous Sarcoma Avian erythrocytosis Avian myeloblastoma Av. erythroblastosis Moloney Sarcoma Kirsten, Harvey sarcoma Finkel Bister Jenkins Feline Sarcoma Simian Sarcoma SOURCE Chicken Chicken chicken chicken mouse rat rat cat monkey ONCOGENE src myb myc erb-A, erb B mos K-ras, H-ras fos fes sis

Slide17: 

What is the origin of transforming genes in sarcoma viruses? How do you prove it?

Origin of Retroviral Transforming Genes: 

Origin of Retroviral Transforming Genes RSV (gag pol env src) r.t. cDNA DENATURE RSV (td: gag,pol,env) isolate genomic RNA gag, pol, env HYBRIDIZE unhybridized sequences hybridized sequences gag, pol, env

Slide19: 

src Origin of Retroviral Transforming Genes Specific src probe HYBRIDIZE RSV-Infected CEF (+ control) “Normal” chick DNA MOUSE DROSOPHILA HUMAN + + + + + Thus: a proto-oncogene is the NORMAL progenitor gene of a viral oncogene

Proto-oncogenes:: 

Proto-oncogenes: J. Michael Bishop and Harold Varmus Nobel Prize: 1989

Retrospective view of the Importance of Retroviral Oncogenes: 

Retrospective view of the Importance of Retroviral Oncogenes While the viral genes might be primarily lab artifacts, they taught us: (1): Proto-oncogenes are highly conserved e.g. mamalian ras genes work perfectly well in Saccharomyces cerevisiae (2): The genes fit into related “families” of related structure and function e.g. src family with conserved PTK domain ras family with conserved guanine nucleotide binding

DEFINITIONS: 

DEFINITIONS ONCOGENE- a gene caried by a virus (RNA or DNA) the expression of which is necessary and sufficient to induce transformation in tissue culture cells and tumors in the appropriate animal PROTO-ONCOGENE-the cellular progenitors of ALL the viral oncogenes e.g., c-K-ras, c-H-ras, c-src, c-myc, etc... AND

PROTO-ONCOGENES:-SOURCES: 

PROTO-ONCOGENES:-SOURCES Genes in the family as viral oncogenes, but not “transduced by viruses- e.g., N-ras, N-myc, L-myc, etc Any gene, which when “activated” can transform NIH-3T3 cells or other immortalized cells- e.g. c-neu, c-met Genes at chromosomal breakpoints- e.g. bcl1, bcl2 Any gene that ain’t covered above that might be important for tumorigenicity or your grant- e.g., int-1

Proto-oncogene:Redefinition: 

Proto-oncogene: Redefinition A DOMINANTLY ACTING, i.e. gain of function gene whose continued activation leads to continued signal transduction, and whose aberrant expression or activity may contribute to tumorigenesis

Evidence for the role of (proto)-oncogenes in malignancies: 

Evidence for the role of (proto)-oncogenes in malignancies Sarcoma Viruses Pathogenesis of leukemia viruses Gene transfer (transfection) Cytogenetics Guilt by association (epidemiology) Chemical Carcinogenesis Radiation Biology Developmental Biology

How Do Leukemia Viruses Cause Leukemia?: 

How Do Leukemia Viruses Cause Leukemia? Leukemia viruses DO NOT contain oncogenes, but DO contain LTRs Although integration of viruses into the host chromosome is relatively random- Tumors are always clonal, and- LTRs are frequently found in proximity to proto-oncogenes

Avian Leukosis Virus Leukemogenesis: 

Avian Leukosis Virus Leukemogenesis ALV ALV Integration Site 5’ or 3’ of gene c-myc gene transcription of myc from LTR ABERRANT REGULATION OF myc TRANSCRIPTION MMTV -int-1 int-2 etc.

Activated Proto-oncogenes from DNA Transfection: 

Activated Proto-oncogenes from DNA Transfection RATIONALE: (1) Viral Oncogenes were not the cause of human tumors (2) Human tumors were (mostly) not caused by activation of any SINGLE gene (3) The genes from human tumors should be sought

Slide29: 

Activated Proto-oncogenes from DNA Transfection Human Bladder Tumor cell line DNA Isolate high MW DNA Isolate DNA fragments restriction endonuclease TRANSFECTION NIH 3T3 fibroblasts TRANSFORMATION Isolate DNA (>99% mouse + 8-10 human genes) TRANSFORMATION Isolate Human DNA

Slide30: 

Activated Proto-oncogenes from DNA Transfection (cont.) RESULT: RAS “Activation” is due to a SINGLE point mutation (gly val) at codon 12 Use Alu probe Isolate Human DNA RESULT: A SINGLE human gene is responsible for transforming capability Sequence RESULT: The gene is the HUMAN c-H-ras gene !!! Compare sequence to NORMAL gene

TRANSFECTION-The Good and the Bad: 

TRANSFECTION- The Good and the Bad It WAS a human Gene But it was still only one gene!!! epidemiology supports multi-hit requirements for most tumors mouse skin carcinogenesis defines specific stages And... It was (mostly) only ras 10-20% of ALL human tumors have activated ras of one kind or another NIH-3T3 cells ain’t exactly normal MORAL: Transfection assays are useful but limited

Transfection of Rat Diploid Embryo Fibroblasts: 

Transfection of Rat Diploid Embryo Fibroblasts + EJ ras FOCI CONCLUSION: At least two genes are required for transformation of “normal” cells

Complementation groups of Proto-oncogenes: 

Complementation groups of Proto-oncogenes ESTABLISHMENT (IMMORTALIZATION) GENES c-myc, N-myc p53* E1A (adenovirus) Large T (polyoma NUCLEAR TRANSFORMING GENES c-K-ras, C-H-ras N-ras c-neu CYTOPLASMIC

The Field Comes Of Age: 

The Field Comes Of Age 1984- Avian erythroblastosis virus gene (v-erbB) is shown to be a truncated Epidermal Growth Factor receptor 1985- Simian sarcoma virus transforming gene (v-sis) is shown to be analogous to the  chain of PDGF receptor 1987- v-jun Avian oncogene is shown to be analogous to AP-1 MORAL: Proto-oncogenes regulate signal transduction WHAT DO THESE CRAZY PROTO-ONCOGENES DO?

Oncogenes in the 21st Century: 

Oncogenes in the 21st Century STI 571 (Gleevec, Imatininb) is the first line therapy for CML The target protein is Bcr-Abl, a protein tyrosine kinase (Abl was first known from a retrovirus) Therapy resistance stems from changes in other proto-oncogenes such as Src Second line and combination therapies with Src inhibitors are becoming standard Src inhibitors entered trial in 2005 BUT-there are failures as well as successes-EGF-R inhibitors are of limited value, so there is still much to learn

Slide36: 

RECEPTOR TYROSINE KINASE GRB-2 SOS RAS RAF MAPKK MEMBRANE MAPK Dimerization; autophosphorylation Interaction of signaling molecules Activation of “downstream” kinases JAK STAT NUCLEUS MAPK Phosphorylation of transcription factors STAT GENE EXPRESSION CYTOPLASM fos, jun, myc SIGNAL TRANSDUCTION

Model for Colon Cancer Development and Progression: 

Model for Colon Cancer Development and Progression After Vogelstein and colleagues (fap) (DCC) (MCC; HNPCC) (p53)